Immunogenic compositions and vaccine compositions comprising biological ingredients, such as viruses, bacteria, parasites, fungi, proteins, polypeptides, glycoproteins, and especially, attenuated live microorganisms, are markedly sensitive to the conditions by which they are prepared, formulated and stored.
Such biological ingredients can be modified and degraded by chemical reactions (e.g. hydrolysis, deamination, Maillard's reaction), many of which are mediated by water. Liquid water allows for molecular movements and can result in modification of protein conformations in compositions comprising biological ingredients. By limiting access to water or by removing water, a major factor of modification and degradation is reduced. Prior methods to confer stability to biological ingredients have primarily involved freezing the water or removing water by freeze-drying.
Lyophilization, or the process of freeze-drying, is a commonly used technique to remove water in the preparation of dehydrated products. Generally, “freeze-drying” an aqueous composition involves three steps. First, the aqueous composition is frozen under conditions of low temperature. Secondly, the frozen water is removed by sublimation under conditions of reduced pressure and low temperature. At this stage, the composition usually contains about 15% water. Third, the residual water is further removed by desorption under conditions of reduced pressure and higher temperatures. At the end of the lyophilization process, a freeze-dried product, also called a “pastille” or “cake” is produced. The freeze-dried product contains very low residual water (from about 0.5% to about 5% weight/weight) and dry material in an amorphous form. This specific state is qualified as “vitreous”.
However, substantial loss of immunogenic activity of biological ingredients are observed during the preparation stages, such as before and during lyophilization, and also during storage of immunogenic compositions and vaccine compositions. The integrity of biological ingredients must be safeguarded to ensure that the immunization efficiency of immunogenic compositions and vaccine compositions is retained. The immunogenic activity of biological ingredients is measured by the ability to induce and stimulate an immunologic response when administered to a host or subject.
To limit the manipulation of subjects and the number of administrations, there is a strong need in the art to provide multivalent immunogenic compositions or vaccine compositions. By definition, a multivalent immunogenic composition or vaccine composition comprises more than one active immunogenic component originating from, or derived from, at least two different pathogens. Viruses from different genera can vary in stability during the freeze-drying step and subsequent storage period, resulting in a loss of viability or infectivity. In the case of viruses such as canine paramyxoviruses, the commonly administered active immunogenic components are live attenuated viruses. To efficiently stimulate the immune system, live attenuated viruses must replicate in the immunized subject. Loss of viability or infectivity can occur during the process of freeze-drying multivalent immunogenic compositions or vaccine compositions, during storage of the compositions, or before administration of the compositions after reconstitution. Thus, stabilizers have been added to such freeze-dried compositions. However, to obtain multivalent immunogenic compositions or vaccine compositions that retain their infectivity and/or viability, a common stabilizer that is able to preserve viability and infectivity of different live attenuated pathogens would be particularly advantageous.
Stabilization of biological ingredients in dry form has typically involved the preservation of antitoxins, antigens and bacteria (Flosodort et al (1935) J. Immunol. 29, 389). However, a limitation in this process included partial denaturation of proteins when dried from an aqueous state at ambient temperatures. Drying from the frozen state helped reduce denaturation and led to better, although incomplete, preservation of biological ingredients including bacteria and viruses (Stamp et al. (1947) J. Gen. Microbiol. 1, 251; Rightsel et al. (1967) Cryobiology 3, 423; Rowe et al. (1971) Cryobiology 8, 251).
More recently, sugars such as sucrose, raffinose and trehalose have been added in various combinations as stabilizers prior to lyophilization of viruses. A large number of compounds have been tested for their ability to stabilize different vaccines containing live attenuated biological ingredients, in particular viruses. Such compounds include SPGA (sucrose, phosphate, glutamate, and albumin; Bovarnick et al. (1950) J. Bacteriol. 59, 509-522; U.S. Pat. No. 4,000,256), bovine or human serum albumin, alkali metal salts of glutamic acid, aluminum salts, sucrose, gelatin, starch, lactose, sorbitol, Tris-EDTA, casein hydrolysate, sodium and potassium lactobionate, and monometallic or dimetallic alkali metal phosphate. Other compounds include, for example, SPG-NZ amine (e.g. U.S. Pat. No. 3,783,098) and polyvinylpyrrolidone (PVP) mixtures (e.g. U.S. Pat. No. 3,915,794). Recently, live attenuated flaviviruses have been stabilized using a complex mixture of multiple compounds, including sorbitol, sucrose, optionally trehalose and/or other disaccharide or trisaccharides, urea, and a specific combination of amino acids (US 2010/0015180A1, to Sanofi Pasteur).
Vaccine and immunogenic compositions have had a tremendous impact on public health by reducing morbidity and mortality from a variety of virulent pathogens. However, unintended side effects arising from additives in immunogenic compositions and vaccine compositions continue to pose a potential risk that may outweigh any protective and therapeutic attributes of immunogenic compositions and vaccine compositions.
In the form frequently used in vaccines in the United States, gelatin can provoke serious allergic reactions in about 1 out of 2 million doses. Allergic reactions previously thought to result from albumin (egg protein) are more likely caused by gelatin in the same vaccine. In the case of human serum albumin, while no disease has ever been associated with human serum albumin in vaccines, there is a chance of transmission of a virus through this protein, which is derived from human blood.
Bovine-derived products, such as bovine albumin and gelatin, carries the risk of transmission of CJD (Creutzfeld-Jakob disease, also known as “Mad Cow Disease”) through beef blood and connective tissue products used in vaccine manufacturing. However, there have been no reported cases where CJD was transmitted through blood or connective tissue products, the prions that cause CJD have not been found in blood or connective tissue, and the use of bovine-derived products from cows imported from countries where there are known cases of Mad Cow Disease is prohibited. Nevertheless, in view of these risks, efforts have been made to eliminate the use of such products in immunogenic compositions that have been observed to elicit unwanted immune effects.
De Rizzo (de Rizzo et al. (1989) Bull. Pan. Am. Health Organ. 23(3), 299-305) reported freeze-dried measles virus preparations containing sorbitol-gelatin or glutamic acid-lactose solutions. These preparations were stored at −20° C. and their viral titers were determined over a 21 month storage period. The resulting data indicated that the freeze-dried viruses without stabilizer are stable when stored at −20° C. over a period of 21 months. Furthermore, it is well known that freeze-dried measles viruses are stable when stored at −20° C. and can retain potency with virtually no loss for many years (Gray A., (1978) Dev. Biol. Stand. 41, 265-266). However, these results were obtained at −20° C. where freeze-dried measles viruses are stable and do not demonstrate an additional stabilizing effect. These results show only that sorbitol-gelatin and glutamic acid-lactose solutions have no negative effect on the stability of the measles viruses that are stored in freeze-dried form at −20° C.
Precausta (Precausta et al (1980) J. Clin. Microbiol. 12(4), 483-489) examined the effects of residual moisture and sealing atmosphere on the infectivity titer of canine distemper virus (CDV) and infectious bronchitis virus (IBV) after freeze-drying. A lactose solution was added to the preparation of CDV to a final concentration of 75 mg/ml, while the IBV vaccine contained 40 mg of mannitol per ml. When CDV titer before freeze-drying was compared to the titer after freeze-drying and after 12 months of storage at 6° C., the CDV titer is decreased from 101.6 to 102.0 CCID50/ml, which reflects a very significant reduction in CDV titer.
Several methods are known in the art for removing moisture for the purpose of preserving biological preparation. The term “spray-freeze-drying” is understood to mean the spraying with a fluid in a cryogenic environment, followed by freeze-drying of the frozen particles obtained. “Foam-drying” is understood to mean the drying, in the form of a glassy foam, by evaporation of water, of a concentrated solution. And “freeze-foam-drying” is intended to mean the drying, in the form of a glassy foam by sublimation of ice, of a pre-frozen solution, at a temperature below the glass transition temperature and the matrix collapse temperature.
Consequently, there is a need for new stabilizers and methods for preserving viability and infectivity of biological ingredients in freeze-dried form, which are safe and suitable for injection to subjects and which have a good aspect.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.